High throughput detection of retrovirus-associated reverse transcriptase using an improved fluorescent product enhanced reverse transcriptase assay and its comparison to conventional detection methods

A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors

“…A number of retrovirus RTs perform similarly in this assay and it is highly quantitative and precise 22,23 (and data not shown).…”

“…The cells are serially passaged for up to 10 passages over a 4 to 5 week period, and the final culture supernatants are assayed using the F-PERT assay. 23 The cutoff point for a positive result in the F-PERT assay has been defined by trend analysis of data by the CRO, and through discussion with the regulatory authorities, as a Ct value of 35.69 or lower. A clinical lot is shown to be free of RCL if the Ct values for the concentrated final passage supernatants from all flasks of the test article are above the cutoff.…”

“…The F-PERT assay is proving to be highly sensitive and reproducible as a means of detecting RT associated with retroviral entities, as has been previously described. 22,23 The culture and amplification process defined by the RCL assay increases the amount of MLV 4070A to approximately three orders of magnitude above the detection limit of the F-PERT assay within the first three or four passages. Therefore, the assay would detect an RCL with highly attenuated replication characteristics, by its amplification above the detection limit at the end of the 4-to 5-week amplification period.…”

“…It is known that the high sensitivity of the F-PERT assay means that occasionally the assay can lead to false positive results, either due to endogenous retroviral RT activity released from the tissue culture cells under stress, or due to cellular polymerases that possess RT-like activity. 23 In such cases, filtered supernatant fluid from the final passage was exposed to fresh HEK293 amplification cells, followed by a two additional passages in order to remove nonreplicating RT activity, and positive and negative controls were included. All cases where RT activity was detected in the end point F-PERT analysis were attributed to non-replicating RT, as the RT activity was not transferred to fresh amplification cells during the additional testing.…”

“…23 For the 'informational' analysis of unconcentrated supernatant fluid samples, a modified F-PERT assay to that previously reported was used. 20,22 Defining the minimal infectious dose of MLV 4070A in HEK293 cells MLV 4070A obtained from the ATCC had a titre determined on PG-4 cells of 1.5 Â 10 7 focus forming units per ml (FFU ml À1 ).…”